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Although the amygdala is known as a negative emotion center for coordinating defensive behaviors, its functions in autonomic control remain unclear. To resolve this issue, we examined effects on cardiovascular responses induced by stimulation and lesions of the amygdala in anesthetized and free-moving rats. Electrical microstimulation of the central nucleus of the amygdala (CeA) induced a gradual increase in arterial pressure (AP) and heart rate (HR), whereas stimulation of adjacent nuclei evoked a phasic AP decrease. The gain of the baroreceptor reflex was not altered by CeA stimulation, suggesting that CeA activity increases both AP and HR by resetting baroreceptor reflex function. Disinhibition of GABAergic input by amygdalar microinjection of the GABA A receptor antagonist induced robust increases in AP and HR. Furthermore, bilateral electrolytic lesions of CeA evoked consistent AP increases over the light/dark cycle. These results suggest that the amygdala exerts 'bidirectional' autonomic control over the cardiovascular system.

The daily rhythm of glucose metabolism is governed by the circadian clock, which consists of cell-autonomous clock machineries residing in nearly every tissue in the body. Disruption of these clock machineries either environmentally or genetically induces the dysregulation of glucose metabolism. Although the roles of clock machineries in the regulation of glucose metabolism have been uncovered in major metabolic tissues, such as the pancreas, liver, and skeletal muscle, it remains unknown whether clock function in non-major metabolic tissues also affects systemic glucose metabolism. Here, we tested the hypothesis that disruption of the clock machinery in the heart might also affect systemic glucose metabolism, because heart function is known to be associated with glucose tolerance. We examined glucose and insulin tolerance as well as heart phenotypes in mice with heart-specific deletion of Bmal1, a core clock gene. Bmal1 deletion in the heart not only decreased heart function but also led to systemic insulin resistance. Moreover, hyperglycemia was induced with age. Furthermore, heart-specific Bmal1-deficient mice exhibited decreased insulin-induced phosphorylation of Akt in the liver, thus indicating that Bmal1 deletion in the heart causes hepatic insulin resistance. Our findings revealed an unexpected effect of the function of clock machinery in a non-major metabolic tissue, the heart, on systemic glucose metabolism in mammals.

Background and Aims: We developed a bio-artificial liver (BAL) using a radial-flow bioreactor and rescued mini-pig models with lethal acute liver failure (ALF). The point of the rescue is the recovery from hepatic encephalopathy (HE). HE on ALF has sometimes resulted in brain death following brain edema with astrocyte swelling. Several factors, including ammonia and glutamine, have been reported to induce astrocyte swelling and injury. However, many clinicians believe that there are any other factors involved in the development of HE. Therefore, the aim of this study was to identify novel HE-inducible factors, particularly those inducing astrocyte dysfunction., Methods: Mini-pig plasma samples were collected at three time points: before the administration of toxins (α-amanitin and LPS), when HE occurred after the administration of toxins, and after treatment with extracorporeal circulation (EC) by the BAL. To identify the causative factors of HE, each plasma sample was subjected to a comparative proteome analysis with two-dimensional gel electrophoresis and mass spectrometry. To assess the direct effects of candidate factors on the astrocyte function and injury, in vitro experiments with human astrocytes were performed., Results: Using a proteome analysis, we identified alpha-1 antichymotrypsin (ACT), which was increased in plasma samples from mini-pigs with HE and decreased in those after treatment with EC by BAL. In in vitro experiments with human astrocytes, ACT showed growth-inhibitory and cytotoxic effects on astrocytes. In addition, the expression of water channel protein aquaporin-4, which is induced in injured astrocytes, was increased following ACT treatment. Interestingly, these effects of ACT were additively enhanced by adding arginine-vasopressin (AVP) and were canceled by adding an AVP receptor antagonist., Conclusions: These results suggest that ACT is involved in astrocyte injury and dysfunction in concert with AVP during the development of acute HE.

The tuberomammillary nucleus (TMN) of the posterior hypothalamus has a high density of histaminergic neurons, the projection fibers of which are present in many areas of the brain, including the nucleus tractus solitarius (NTS), which controls arterial pressure (AP). In this study, we investigated whether the TMN-NTS pathway is involved in central cardiovascular regulation. Bicuculline, a gamma-aminobutyric acid type A (GABA A ) receptor antagonist, was microinjected into the ventral TMN of anesthetized rats and its effects on AP and heart rate (HR) were observed. We also evaluated the effect of cetirizine, an H 1 receptor antagonist, microinjected into the NTS on cardiovascular responses induced by electrical stimulation of the TMN Both AP and HR increased following bicuculline microinjection into the ventral TMN Similar pressor and tachycardic responses were observed after electrical stimulation of the ventral TMN Microinjection of cetirizine into the NTS partially inhibited the pressor response but had no effect on HR Finally, the treadmill test was associated with a high level of c-Fos expression in both ventral TMN and NTS neurons. These results suggest that the TMN-NTS pathway is involved in regulation of AP, presumably under a high-arousal phase, such as that during exercise., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Aim: Our previous findings suggest that the nucleus of the solitary tract (NTS), a pivotal region for regulating the set point of arterial pressure, exhibits abnormal inflammation in pre-hypertensive and spontaneously hypertensive rats (SHRs), with elevated anti-apoptotic and low apoptotic factor levels compared with that of normotensive Wistar-Kyoto (WKY) rats. Whether this chronic condition affects neuronal growth and plasticity in the NTS remains unknown. To unveil the characteristics of the neurodevelopmental environment in the NTS of SHRs, we investigated the expression of neurotrophic factors transcripts in SHRs., Methods: RT(2) Profiler PCR Array targeting rat neurotrophins and their receptors was used to screen for differentially expressed transcripts in the NTS of SHRs compared to that of WKY rats. Protein expression and physiological functions of some of the differentially expressed transcripts were also studied., Results: Gene and protein expressions of glial cell line-derived neurotrophic factor family receptor alpha-3 (Gfrα-3) factor were both upregulated in the NTS of adult SHRs. Gene expressions of corticotropin-releasing hormone-binding protein (Crhbp), interleukin-10 receptor alpha (Il-10ra) and hypocretin (Hcrt) were downregulated in the NTS of adult SHRs. The Gfrα-3 transcript was increased and the Hcrt transcript was decreased in the NTS of young pre-hypertensive SHRs, suggesting that these profiles are not secondary to hypertension. Moreover, microinjection in the NTS of hypocretin-1 decreased blood pressure in adult SHRs., Conclusion: These results suggest that altered neurotrophic factors transcript profiles may affect the normal development and function of neuronal circuitry that regulates cardiovascular autonomic activity, thereby resulting in manifestations of neurogenic hypertension in SHRs., (© 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Cardiac function is highly dependent on oxidative energy, which is produced by mitochondrial respiration. Defects in mitochondrial function are associated with both structural and functional abnormalities in the heart. Here, we show that heart-specific ablation of the circadian clock gene Bmal1 results in cardiac mitochondrial defects that include morphological changes and functional abnormalities, such as reduced enzymatic activities within the respiratory complex. Mice without cardiac Bmal1 function show a significant decrease in the expression of genes associated with the fatty acid oxidative pathway, the tricarboxylic acid cycle, and the mitochondrial respiratory chain in the heart and develop severe progressive heart failure with age. Importantly, similar changes in gene expression related to mitochondrial oxidative metabolism are also observed in C57BL/6J mice subjected to chronic reversal of the light-dark cycle; thus, they show disrupted circadian rhythmicity. These findings indicate that the circadian clock system plays an important role in regulating mitochondrial metabolism and thereby maintains cardiac function.

Aim: The nucleus tractus solitarii (NTS) is a central brainstem structure that plays an important role in regulating cardiovascular homeostasis. In this study, we examined whether H1 receptors in the NTS can control the baroreceptor reflex function by modulating synaptic transmission., Methods: Cardiac baroreceptor reflex function was assessed before and after the microinjection of 2-pyridylethylamine (10-25 nmol), a histamine H1 receptor-specific agonist, into the NTS of urethane-anaesthetized Wistar rats. The cardiovascular responses induced by l-glutamate microinjection into the NTS were also examined before and after the NTS administration of 2-pyridylethylamine., Results: Nucleus tractus solitarii microinjections of 2-pyridylethylamine significantly inhibited the gain of the cardiac baroreceptor reflex and bradycardiac/depressor responses induced by l-glutamate microinjection into the NTS. These findings suggest that histamine H1 receptors regulate the cardiac baroreceptor reflex in a post-synaptic manner to inhibit barosensitive NTS neurons., Conclusion: Taken together with our previous findings, the present results provide further evidence that histamine may play a role within the NTS in regulating cardiovascular homeostasis., (© 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

The nucleus tractus solitarii (NTS) controls the cardiovascular system during exercise, and alteration of its function may underlie exercise-induced cardiovascular adaptation. To understand the molecular basis of the NTS's plasticity in regulating blood pressure (BP) and its potential contribution to the antihypertensive effects, we characterized the gene expression profiles at the level of the NTS after long-term daily wheel running in spontaneously hypertensive rats (SHRs). Genome-wide microarray analysis was performed to screen for differentially expressed genes in the NTS between exercise-trained (12 wk) and control SHRs. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes database revealed that daily exercise altered the expression levels of NTS genes that are functionally associated with metabolic pathways (5 genes), neuroactive ligand-receptor interactions (4 genes), cell adhesion molecules (3 genes), and cytokine-cytokine receptor interactions (3 genes). One of the genes that belonged to the neuroactive ligand-receptor interactions category was histamine receptor H(1). Since we confirmed that the pressor response induced by activation of this receptor is increased after long-term daily exercise, it is suggested that functional plasticity in the histaminergic system may mediate the facilitation of blood pressure control in response to exercise but may not be involved in the lowered basal BP level found in exercise-trained SHRs. Since abnormal inflammatory states in the NTS are known to be prohypertensive in SHRs, altered gene expression of the inflammatory molecules identified in this study may be related to the antihypertensive effects in exercise-trained SHRs, although such speculation awaits functional validation.

The magnocellular neurones (MCN) of the supraoptic nucleus (SON) undergo reversible changes during dehydration. We hypothesise that alterations in steady-state transcript levels might be partially responsible for this plasticity. In turn, regulation of transcript abundance might be mediated by transcription factors. We have previously used microarrays to identify changes in the expression of mRNAs encoding transcription factors in response to water deprivation. We observed down-regulation of 11 and up-regulation of 31 transcription factor transcripts, including members of the activator protein-1 gene family, namely c-fos, c-jun, fosl1 and junD. Because JunD expression and regulation within the SON has not been previously described, we have used in situ hybridisation and the quantitative reverse transcriptase-polymerase chain reaction to confirm the array results, demonstrating a significant increase in JunD mRNA levels following 24 and 72 h of water deprivation. Western blot and immunohistochemistry revealed a significant increase in JunD protein expression following dehydration. Double-staining fluorescence immunohistochemistry with a neurone-specific marker (NeuN) demonstrated that JunD staining is predominantly neuronal. Additionally, JunD immunoreactivity is observed primarily in vasopressin-containing neurones with markedly less staining seen in oxytocin-containing MCNs. Furthermore, JunD is highly co-expressed with c-Fos in MCNs of the SON following dehydration. These results suggest that JunD plays a role in the regulation of gene expression within MCNs of the SON in association with other Fos and Jun family members., (© 2012 The Authors. Journal of Neuroendocrinology © 2012 British Society for Neuroendocrinology.)

Understanding how the 24-hour blood-pressure rhythm is programmed has been one of the most challenging questions in cardiovascular research. The 24-hour blood-pressure rhythm is primarily driven by the circadian clock system, in which the master circadian pacemaker within the suprachiasmatic nuclei of the hypothalamus is first entrained to the light/dark cycle and then transmits synchronizing signals to the peripheral clocks common to most tissues, including the heart and blood vessels. However, the circadian system is more complex than this basic hierarchical structure, as indicated by the discovery that peripheral clocks are either influenced to some degree or fully driven by temporal changes in energy homeostasis, independent of the light entrainment pathway. Through various comparative genomic approaches and through studies exploiting mouse genetics and transgenics, we now appreciate that cardiovascular tissues possess a large number of metabolic genes whose expression cycle and reciprocally affect the transcriptional control of major circadian clock genes. These findings indicate that metabolic cycles can directly or indirectly affect the diurnal rhythm of cardiovascular function. Here, we discuss a framework for understanding how the 24-hour blood-pressure rhythm is driven by the circadian system that integrates cardiovascular and metabolic function.

Essential hypertension is idiopathic although it is accepted as a complex polygenic trait with underlying genetic components, which remain unknown. Our supposition is that primary hypertension involves activation of the sympathetic nervous system. One pivotal region controlling arterial pressure set point is nucleus tractus solitarii (NTS). We recently identified that pro-inflammatory molecules, such as junctional adhesion molecule-1, were over expressed in endothelial cells of the microvasculature supplying the NTS in an animal model of human hypertension (the spontaneously hypertensive rat: SHR) compared to normotensive Wistar Kyoto (WKY) rats. We have also shown endogenous leukocyte accumulation inside capillaries within the NTS of SHR but not WKY rats. Despite the inflammatory state in the NTS of SHR, transcripts of some inflammatory molecules such as chemokine (C-C motif) ligand 5 (Ccl5), and its receptors, chemokine (C-C motif) receptor 1 and 3 were down-regulated in the NTS of SHR compared to WKY rats. This may be compensatory to avoid further strong inflammatory activity. More importantly, we found that down-regulation of Ccl5 in the NTS of SHR may be pro-hypertensive since microinjection of Ccl5 into the NTS of SHR decreased arterial pressure but was less effective in WKY rats. Leukocyte accumulation of the NTS microvasculature may also induce an increase in vascular resistance and hypoperfusion within the NTS; the latter may trigger release of pro-inflammatory molecules which via paracrine signaling may affect central neural cardiovascular activity conducive to neurogenic hypertension. All told, we suggest that vascular inflammation within the brainstem contributes to neurogenic hypertension by multiple pathways., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Axons of histamine (HA)-containing neurons are known to project from the posterior hypothalamus to many areas of the brain, including the nucleus tractus solitarii (NTS), a central brain structure that plays an important role in regulating arterial pressure. However, the functional significance of NTS HA is still not fully established. In this study, we microinjected HA or 2-pyridylethylamine, a HA-receptor H(1)-specific agonist, into the NTS of urethane-anesthetized Wister rats to identify the potential functions of NTS HA on cardiovascular regulation. When HA or H(1)-receptor-specific agonist was bilaterally microinjected into the NTS, mean arterial pressure (MAP) and heart rate (HR) were significantly increased, whereas pretreatment with the H(1)-receptor-specific antagonist cetirizine into the NTS significantly inhibited the cardiovascular responses. The maximal responses of MAP and HR changes induced by HA or H(1)-receptor-specific agonist were dose dependent. We also confirmed gene expression of HA receptors in the NTS and that the expression level of H(1) mRNA was higher than that of the other subtypes. In addition, we found that H(1) receptors are mainly expressed in neurons of the NTS. These findings suggested that HA within the NTS may play a role in regulating cardiovascular homeostasis via activation of H(1) receptors expressed in the NTS neurons.

Since the nucleus tractus solitarii (NTS) is a pivotal region for regulating the set-point of arterial pressure, we propose here its role in the development of neurogenic hypertension. Given the findings of recent studies suggesting that the NTS of spontaneously hypertensive rats (SHR) exhibits a specific inflammatory state characterized by leukocyte accumulation within the NTS microvasculature, we hypothesized that gene expression levels of apoptotic factors are altered in the NTS of SHR compared to normotensive Wistar-Kyoto rats (WKY). To test this hypothesis, we used RT(2) Profiler PCR arrays targeting apoptosis-related factors. We found that gene expression of the death receptor Fas (tumor necrosis factor receptor superfamily, member 6) and the cysteine-aspartic acid protease caspase 12 were down-regulated in the NTS of both adult hypertensive and young pre-hypertensive SHR compared to age-matched WKY. On the other hand, an anti-apoptotic factor, neuronal apoptosis inhibitory protein, was highly increased in the NTS of SHR. These results suggest that the NTS of SHR exhibits an anti-apoptotic condition. Furthermore, this profile appears not to be secondary to hypertension. Whether this differential gene expression in the NTS contributes to the hypertensive state of the SHR via alteration of neuronal circuitry regulating cardiovascular autonomic activity awaits elucidation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: The loss of diurnal rhythm in blood pressure (BP) is an important predictor of end-organ damage in hypertensive and diabetic patients. Recent evidence has suggested that two major physiological circadian rhythms, the metabolic and cardiovascular rhythms, are subject to regulation by overlapping molecular pathways, indicating that dysregulation of metabolic cycles could desynchronize the normal diurnal rhythm of BP with the daily light/dark cycle. However, little is known about the impact of changes in metabolic cycles on BP diurnal rhythm., Methodology/principal Findings: To test the hypothesis that feeding-fasting cycles could affect the diurnal pattern of BP, we used spontaneously hypertensive rats (SHR) which develop essential hypertension with disrupted diurnal BP rhythms and examined whether abnormal BP rhythms in SHR were caused by alteration in the daily feeding rhythm. We found that SHR exhibit attenuated feeding rhythm which accompanies disrupted rhythms in metabolic gene expression not only in metabolic tissues but also in cardiovascular tissues. More importantly, the correction of abnormal feeding rhythms in SHR restored the daily BP rhythm and was accompanied by changes in the timing of expression of key circadian and metabolic genes in cardiovascular tissues., Conclusions/significance: These results indicate that the metabolic cycle is an important determinant of the cardiovascular diurnal rhythm and that disrupted BP rhythms in hypertensive patients can be normalized by manipulating feeding cycles.

Since the nucleus tractus solitarii (NTS) is a pivotal region for regulating the set-point of arterial pressure, we proposed a role for it in the development of neurogenic hypertension. Recent studies have suggested that proinflammatory molecules, such as junctional adhesion molecule 1 (JAM-1) are highly expressed in the NTS of an animal model of human essential hypertension, the spontaneously hypertensive rat (SHR), compared with normotensive rats (Wistar-Kyoto, WKY). Moreover, we have also shown endogenous leukocyte accumulation inside capillaries within the NTS of SHR but not WKY rats. Based on this evidence, we hypothesized that gene expression of cytokines/chemokines is altered in the NTS of SHR. We have screened for abnormally expressed inflammatory mediators in the NTS of SHR using the RT2 Profiler PCR arrays, which were designed specifically to target major cytokines/chemokines. The specific PCR array revealed that seven genes were less expressed in the NTS of SHR compared with WKY rats (more than twofold differences), while only two genes were more expressed in the SHR. Moreover, we identified that some of these validated molecules exhibit important functional roles for cardiovascular homeostasis at the level of the NTS. We suggest that abnormal gene expression of proinflammatory molecules, such as JAM-1, causes leukocyte accumulation within the vasculature in the NTS of SHR. Consequently, gene expression of specific cytokines/chemokines may be downregulated to avoid further strong inflammatory activity in the NTS of SHR at the expense of an alteration in neuronal activity that leads to cardiovascular autonomic pathology. Importantly, to allow translation of our work, these novel insights need to be assessed in hypertensive human brainstem tissue; their confirmation could lead to novel therapeutic approaches for one of the world's most prevalent diseases.

Recent gene array and molecular studies have suggested that an abnormal gene expression profile of interleukin-6 (IL-6) in the nucleus tractus solitarii (NTS), a pivotal region for regulating arterial pressure, may be related to the development of neurogenic hypertension. However, the precise functional role of IL-6 in the NTS remains unknown. In the present study, we have tested whether IL-6 affects cardiovascular control at the level of the NTS. IL-6 (1, 10, and 100 fmol) was microinjected in the NTS of Wistar rats (280-350 g) under urethane anesthesia. Although the baseline levels of arterial pressure and heart rate did not change following IL-6 injections, the cardiac baroreflex in response to increased arterial pressure was dose-dependently attenuated. In addition, IL-6 (100 fmol) microinjections also attenuated l-glutamate-induced bradycardia at the level of the NTS. Immunohistochemical detection of IL-6 in naïve rats demonstrated that it was predominantly observed in neurons within the brain stem, including the NTS. These findings suggest that IL-6 within the NTS may play an important role for regulating cardiovascular control via modulation of input signals from baroreceptor afferents. Whether the abnormal gene expression of IL-6 in the NTS is associated in a causal way with hypertension remains to be resolved.

Although both alpha(1)- and alpha(2)-adrenergic receptors (ARs) are known to be expressed in the nucleus of the solitary tract (NTS), the functional significance of these receptors is still not fully established. In this study, we microinjected alpha(1)- and alpha(2)-AR agonists into the NTS of urethane-anaesthetized Wister rats to study the cardiovascular effects in response to their activation. When the alpha(1)-AR agonist phenylephrine was microinjected into the area where barosensitive neurons are dominantly located (baro-NTS), mean arterial pressure (MAP) and heart rate (HR) were significantly elevated. When tested in the area where chemosensitive neurons are dominantly located (chemo-NTS), however, MAP and HR were significantly decreased. Pretreatment with the non-specific alpha-AR antagonist phentolamine into the NTS inhibited the phenylephrine-induced cardiovascular responses. In contrast, microinjection of the alpha(2)-AR agonist clonidine into either the baro-NTS or the chemo-NTS decreased MAP and HR; they were also inhibited by the alpha(2)-adrenergic antagonist yohimbine. Moreover, we immunohistochemically identified that cardiovascular responses induced by alpha(1)-ARs may be mediated by NTS neurons while those induced by alpha(2)-ARs may be mediated by astrocytes located in the barosensitive and chemosensitive areas of the NTS. These results suggest that both types of alpha-AR expressed in the NTS may be involved in regulating cardiovascular homeostasis via modulation of input signals from baroreceptor and chemoreceptor afferents; however, cardiovascular responses produced by stimulation of alpha(1)-ARs are strictly location specific within the NTS.

Human essential hypertension is a complex polygenic trait with underlying genetic components that remain unknown. Since the brainstem structure--the nucleus of the solitary tract (NTS)--is a pivotal region for regulating the set-point of arterial pressure, we proposed a role for it in the development of primary hypertension. Using microarray and real-time RT-PCR, we have recently identified that some pro-inflammatory molecules, such as junctional adhesion molecule-1 (JAM-1; a leukocyte/platelet adhesion molecule), were over expressed in endothelial cells in the NTS of an animal model of human essential hypertension--the spontaneously hypertensive rat (SHR) compared to normotensive Wistar Kyoto rats (WKY). Adenoviral mediated over expression of JAM-1 in NTS of WKY rats produced both hypertension and localized leukocyte adherence to the microvasculature. With a known effect of leukocyte adhesion causing cytokine release, we predicted differences in the level of gene expression of cytokines in the NTS of SHR relative to WKY. Gene expression of monocyte chemoattractant protein-1 (MCP-1) was higher in the NTS of SHR while inter-leukin-6 (IL-6) was lower in the NTS of SHR compared to the WKY. Because both inflammatory molecules are known to affect neural functions, our data suggest that the microvasculature of NTS of the SHR exhibits a specific inflammatory state. We propose a new hypothesis that as a consequence of enhanced expression of leukocyte adhesion molecules within the microvasculature of NTS there is a specific inflammatory response that leads to cardiovascular autonomic dysfunction contributing to neurogenic hypertension.

Since the nucleus of the solitary tract (NTS) is a pivotal region for regulating the set-point of arterial pressure, we proposed a role for it in the development of neurogenic hypertension. Recent studies have suggested that pro-inflammatory molecules are highly expressed in the NTS of an animal model of human essential hypertension--the spontaneously hypertensive rat (SHR), compared to normotensive Wistar-Kyoto rat (WKY). Based on this evidence, we hypothesized that inflammatory mediators such as cytokines are up-regulated in the hypertensive NTS. In the present study, we have assessed the level of gene expression of some cytokines in the NTS of SHR compared to WKY. In addition, for further confirmation of abnormal inflammatory condition within the NTS of SHR, we identified gene expression levels of an inflammatory marker, glycoprotein-39 (gp39) precursor, which is homologous to chitinase 3-like protein 1, human cartilage-gp39 or YKL40. The NTS was micro-dissected from 15-week-old male SHR and WKY rats. Total RNA was extracted and quantitative RT-PCR was performed. Gene expression of gp39 precursor and monocyte chemoattractant protein-1 were higher in the NTS of SHR while inter-leukin-6 was lower in the NTS of SHR compared to the WKY. In contrast, there were no significant differences in the expression of other cytokines including: inter-leukin-1 beta, tumor necrosis factor-alpha and transforming growth factor beta 1. These data together with our previous published finding of an over expression of junctional adhesion molecule-1 suggest that the NTS of the SHR exhibits a specific inflammatory state.

The hypothalamic-neurohypophyseal system (HNS) mediates neuroendocrine responses to dehydration through the actions of the antidiuretic hormone vasopressin (VP) and the natriuetic peptide oxytocin (OT). VP and OT are synthesised as separate prepropeptide precursors in the cell bodies of magnocellular neurones in the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus, the axons of which innervate the posterior pituitary gland (PP). Dehydration evokes a massive release of both peptides into the circulation, and this is accompanied by a function-related remodelling of the HNS. Microarray studies on mRNAs differentially expressed in the SON revealed that transcripts encoding the Ywhag and Ywhaz isoforms of the 14-3-3 family of regulatory proteins, are increased in the rat SON by 3 days of water deprivation; findings that we have confirmed by the real-time polymerase chain reaction. Because there is no necessary proportionality between transcript and protein abundance, we next examined Ywhag and Ywhaz translation products throughout the HNS in parallel with 14-3-3 post-translational modification, which is known to be an important determinant of functional activity. Both proteins are robustly expressed in the SON in VP- and OT-containing neurones, but the abundance of neither changes with dehydration. However, the total level of Ywhaz protein is increased in the neurointermediate lobe of the pituitary (NIL, which includes the PP), in parallel with a basic post-translationally modified isoform, suggesting transport from the cell bodies of the SON of newly-synthesised protein and changes in its activity. The level of an acidic, probably phosphorylated, Ywhag isoform is down-regulated in the SON by dehydration, although total levels are unchanged. Finally, based on the presence of a phosphorylated 14-3-3 binding motif, we have identified a 14-3-3 binding partner, proteasome subunit, beta type 7, in the NIL. Thus, we suggest that, through complex transcriptional, and post-translational processes, 14-3-3 proteins are involved in the regulation or mediation of HNS plasticity following dehydration.

The hypothalamo-neurohypophyseal system (HNS) mediates neuroendocrine responses to dehydration through the action of the antidiuretic hormone vasopressin (VP). VP is synthesized as part of a prepropeptide in magnocellular neurons of the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus. This precursor is processed during transport to axon terminals in the posterior pituitary gland, in which biologically active VP is stored until mobilized for secretion by electrical activity evoked by osmotic cues. During release, VP travels through the blood stream to specific receptor targets located in the kidney in which it increases the permeability of the collecting ducts to water, reducing the renal excretion of water, thus promoting water conservation. The HNS undergoes a dramatic function-related plasticity during dehydration. We hypothesize that alterations in steady-state protein levels might be partially responsible for this remodeling. We investigated dehydration-induced changes in the SON and pituitary neurointermediate lobe (NIL) proteomes using two-dimensional fluorescence difference gel electrophoresis. Seventy proteins were altered by dehydration, including 45 in the NIL and 25 in the SON. Using matrix-assisted laser desorption/ionization mass spectrometry, we identified six proteins in the NIL (four down, two up) and nine proteins in the SON (four up, five down) that are regulated as a consequence of chronic dehydration. Results for five of these proteins, namely Hsp1alpha (heat shock protein 1alpha), NAP22 (neuronal axonal membrane protein 22), GRP58 (58 kDa glucose regulated protein), calretinin, and ProSAAS (proprotein convertase subtilisin/kexin type 1 inhibitor), have been confirmed using independent methods such as semiquantitative Western blotting, two-dimensional Western blotting, enzyme-linked immunoassay, and immunohistochemistry. These proteins may have roles in regulating and effecting HNS remodeling.